Glaucoma is the most prevalent optic neuropathy where a progressive degeneration of retinal ganglion cells (RGCs) leads to vision loss. Our long-term goal is to help prevent the degeneration of glaucomatous RGCs by characterizing induced-pluripotent stem cells (iPSCs) as a renewable source of retinal progenitors for autologous ex vivo cell therapy. The objective of this application is to optimize the use of limbal iPSCs to generate RGCs that are functional, safe, and practical for clinical use. The central hypothesis of the proposed study is that the molecular mechanism underlying RGC differentiation is active in iPSC-derived retinal progenitors and recruited in response to specific extrinsic cues to generate RGCs with target specificity. Our reasoning is based on the following observations:(1) retinal progenitors can be derived from limbal iPSCs, generated through safe non-nucleic acid method (2) iPSC-derived retinal progenitors respond to cues conducive for RGC differentiation, and (3) iPSC-derived RGCs demonstrate target specificity. The rationale for the proposed research is that once conditions are identified, we can efficiently generate RGC precursors to treat RGC degeneration through transplantation, and develop a robust model system for testing drugs and genetic approaches for optic neuropathy. Based on our preliminary data the following specific aims are proposed to test the hypothesis: Specific Aim 1: To determine the conditions for generating retinal progenitors from iPSCs, Specific Aim 2: To determine conditions for the generation of RGCs from iPSC-derived retinal progenitors, and Specific Aim 3: To determine the target specificity and in vivo differentiation of iPSC-derived RGCs. The retinal potential will be examined in limbal iPSCs generated by non-nucleic acid means, pioneered in our lab. This approach of reprogramming by recruiting endogenous pluripotency genes instead of introducing exogenous genes, which can lead to insertional mutagenesis, addresses a significant barrier to iPSC-based therapy. Controls will include limbal iPSC derived by a conventional nucleic acid method to compare the effects of two different approaches of reprogramming on the acquisition of retinal and RGC potential. The induction of iPSCs along a neural lineage, their subsequent specification into retinal progenitors, and their final differentiation into RGCs will be accomplished non-cell autonomously by perturbing specific signaling pathways to recapitulate developmental mechanism. Therefore, our research proposed is innovative because it presents an entirely different and a safe approach for reprogramming somatic cells to a pluripotent state and generating RGCs without using nucleic acids or forced expression of exogenous factors. The emerging information will be significant because it will not only address each of the barriers that currently make the ex-vivo stem cell therapy approach impractical but also lead to the development of a robust model system for testing normal mechanisms of RGC development and for screening drugs and genes for additional new approaches for addressing glaucomatous retinal degeneration.